1. Field of the Invention
The present invention relates to the synthesis of a linear, double-stranded, covalently closed DNA molecule that can be used as a vector for gene therapy. More generally, the present invention also relates to a method of obtaining preparations of DNA molecules essentially free of contamination by genomic DNA originating from organisms employed in the making of the DNA molecules.
2. Background Information
Gene therapy and genetic vaccination are modern molecular approaches promising to change the way of future medical practice. Despite the great expectations inspired by these methods, however, some basic problems will have to be solved before these methods find general clinical acceptance.
Both gene therapy and genetic vaccination need methods to transfer genetic information into cells or tissues of a patient and to subsequently express the transferred information within those cells. There are safety, efficacy, and specificity issues associated with this transfer process and the transfer means employed. Generally speaking, viral transfer means are very efficient and specific but may be a cause for concern regarding both epidemiological and immunological issues. Non-viral transfer means such as naked DNA are considered to be much safer regarding possible reversion to pathogenicity, but may offer less efficacy and specificity. These issues are discussed, and some possible solutions offered, in our application WO 98/21322, the disclosure of which is incorporated herein by reference. The main aspect of Application No. WO 98/21322 is a minimalistic vector construct consisting mainly of linear double-stranded DNA, which is covalently closed by short oligodesoxyribonucleotide loops to essentially prevent exonucleolytic degradation and to allow attachment of specificity-inducing moieties. This construct will be referred to within the present specification also as, for example, a dumbbell expression construct, a dumbbell construct, and/or a dumbbell-shaped construct.
Several methods are known or can be fashioned without inventive activity in order to make such constructs. One can amplify the expression cassette forming the main, double-stranded part of the dumbbell construct by polymerase chain reaction (PCR), subsequently digest the amplified fragment by means of restriction enzymes, leaving overlapping ends, and ligate short hairpin oligonucleotides to the ends of the digested amplification fragments. Thereby, one will obtain dumbbell-shaped constructs that are essentially easily purified by HPLC on a large scale. The use of heat-stable or thermostable polymerases, however, makes the process uneconomical and gives rise to impurities in the product due to the high error rate of the polymerase. Alternatively, the main part of the construct can be amplified as part of a bacterial plasmid by fermentation, cut out from the plasmid backbone by restriction digest, and be ligated to hairpin oligonucleotides as described above. This leaves the desired product contaminated by backbone sequences, the removal of which is one main objective of making the minimalistic dumbbell constructs described in the present application. The backbone contamination can be removed by chromatography, electrophoresis, or other methods based on size. Since both vector backbone and expression cassette may be, and in practice often are, in the same size rangexe2x80x94that is, between approximately 1.5 kilobasepairs and approximately 5 kilobasepairs, this separation based on size differences can be difficult and often renders suboptimal results. Therefore, methods are needed to obtain the desired constructs in an essentially easy, economical process.
One object of the present invention may be to provide a process to obtain linear double-stranded covalently closed DNA xe2x80x9cdumbbellxe2x80x9d constructs from plasmids by restriction digest, subsequent ligation with hairpin oligodesoxyribonucleotides, optionally in the presence of restriction enzyme, and a final digest with endo- and exonucleolytic enzymes that degrade essentially all contaminating polymeric DNA molecules but the desired construct. Another object of the present invention may be to provide a process to obtain said dumbbell constructs employing endonuclease class II enzymes, recognizing non-palindromic sequences, and having the restriction site generate overlapping ends away from the enzyme recognition site. Furthermore, yet another object of the invention may be to provide a process to obtain linear, covalently closed DNA molecules, such as plasmids, essentially free from contamination by genomic DNA, by submitting the DNA preparation to a facultative endonucleolytic degradation step and an obligatory exonucleolytic degradation step.
According to the present invention, the DNA molecule forming the main, double-stranded part of the desired expression cassette dumbbell construct may be amplified by fermentation as part of a bacterial plasmid. The DNA molecule is isolated from the plasmid, on or in which it can be contained as a single or multiple copy within the plasmid sequence. Subsequently, the DNA molecule may be cut from the vector backbone by restriction endonucleases that may leave essentially short single overlaps at the restriction ends, preferably of three or more nucleotides in length.
In the next step, the resulting mix of expression cassette construct and vector backbone may be reacted in the presence of a DNA ligase with essentially short, hairpin-forming oligodesoxyribonucleotides that may comprise a single-stranded overlap hybridizing to the overlap generated by the restriction enzyme, resulting in a covalently closed single-stranded molecule with oligonucleotide loops at both ends. According to the invention, the resulting mix of dumbbell-shaped expression construct and backbone sequences, at least some of which backbone sequences may be in dumbbell form also, may be reacted with a restriction endonuclease that cuts only the backbone sequence and the recognition sequence that is not provided on the dumbbell expression construct. This endonuclease digest renders a mix of covalently closed molecules of the desired product, as well as digested backbone molecules and contaminating sequences with open 5xe2x80x2 hydroxyl ends and 3xe2x80x2 hydroxyl ends. This mixture is subsequently submitted to extensive exonuclease digestion. The use of the exonuclease activity of bacteriophage T4 or T7 DNA polymerases was found, in at least one embodiment of the present invention, to be the best mode of executing this step of exonuclease digestion because of the essentially high specificity and processivity of these enzymes. However, any other specifically exonucleolytic activity can be employed to practice at least one possible embodiment of the present invention. The resulting digestion product is a mixture of the desired dumbbell construct, enzymes and buffer components, and desoxynucleotide monomers. From this mixture, the desired product can be purified essentially easy in a single and simple chromatographic step.
The main class of restriction enzymes used in molecular biology is endonuclease class I. These enzymes recognize short palindromic sequences and cut within the recognition site. These enzymes give very practical results when the products are to be used compatibly in cloning experiments. In the ligation described above, however, they may offer a serious drawback. The palindromic overlap generated by the enzyme can lead to reactions between restriction fragments, which reactions are referred to in the present specification as intra- or inter-polymeric reactions. The desired reaction, however, is a polymer-to-oligomer reaction. The former type of reactionxe2x80x94that is, intra- or inter-polymeric reactionsxe2x80x94can be suppressed by an essentially large excess of hairpin oligomer, which in turn leads to the formation of hairpin dimers.
According to another aspect of the invention, this problemxe2x80x94that is, the problem of intra- or inter-polymeric reactions between or among restriction fragmentsxe2x80x94can be avoided in at least two ways. Firstly, hybridizing overlaps from two different recognition sites can be employed that do not generate, reconstitute, or reconstruct a palindromic sequence upon reaction between a polymer and an oligomer molecule, but instead may regenerate the in situ respective recognition site if hairpin dimers or polymeric dimers are formed. Both latter speciesxe2x80x94that is, both hairpin dimers and polymeric dimersxe2x80x94may be re-digested in the presence of ligase. Because hairpin-polymer ligation products do not form a palindromic site and are not re-digested, they may accumulate during the course of the reaction. One example of this strategy is the usage of an EcoRI site (Gxe2x80x2AATTC, where the symbol xe2x80x9c{acute over ( )}xe2x80x9d signifies the cut site, overlap AATT) to cut out or cleave the desired sequence from the plasmid and the use of an AATTG sequence for the 5xe2x80x2 end of the hairpin, where AATT is the overlap sequence and G the first base of the double-stranded stem. Ligation of the plasmid fragment and the oligonucleotide may render or result in a ligation product with sequence GAATTG, which is not cut by any class I enzyme. If two ends of the plasmid religate, they can, in at least one possible embodiment of the present invention, be re-cut by EcoRI in situ, and an oligo dimer can be re-digested by MunI in situ (recognition sequence CAATTG).
Secondly, according to another aspect of the invention, the aforementioned problem of the need to employ a large excess of hairpin oligomer can be avoided by employing class II restriction endonucleases, which recognize non-palindromic sequences and cut away from their recognition site (see Butkus et al., xe2x80x9cA New Restriction Endonuclease Eco31I Recognizing a Non-Palindromic Sequence,xe2x80x9d Biochim. Biophys. Acta Dec. 18, 1985; 826(4): 208-12, which publication is hereby incorporated by reference into this application). Many of these enzymes can be obtained from numerous commercial sources (for example, MBI-Fermentas, Vilnius, Lithuania, and New England Biolabs, Massachusetts, USA). The resulting ligation strategy employs hairpin oligonucleotides with a non-palindromic overlap. These molecules cannot ligate with one another and thus their active concentration in the ligation reaction diminishes through ligation to the vector fragment only. Consequently, the hairpin concentration leading to essentially exclusively monomeric fragment dumbbell products is much lower than the concentration resulting from employing palindromic sequences. Further improvement of the product quality can be achieved by adding a class II restriction enzyme to the ligation reaction in situ, which leads to re-digestion of inadvertently formed dimers.
According to yet another aspect of the invention, the digestion with a nonspecific exonuclease of preparations of covalently closed DNA obtained by fermentation can be used to purify such DNA preparations. This achieves an essentially great improvement over existing methods of purifying biotechnologically obtained DNA. The quality specifications given by one leading provider of so-called GMP-grade DNA give as release criteria the content of xe2x80x9chost DNA  less than 5%xe2x80x9d (source: online catalog of Qiagen GmbH, which catalog is hereby incorporated by reference into this specification, entry as of Jun. 11, 1999, having the following URL or Internet link: http://www.giagen.com/catalog/chapter 12/chap 12b.asp). It may be expected that such an essentially high degree of contamination will be the cause of calls for improvement.
Therefore, the present invention also may extend to a purification process for a preparation of any covalently closed DNA, such as a plasmid, derived from fermentative processes in prokaryotic or eukaryotic hosts. In at least one possible embodiment of the present invention, this process entails a digestion step using an exonuclease. Additionally, endonucleases can be added to digest the host genomic DNA; these endonucleases must not find an operable recognition site on the molecule to be purified. Preferably, a plurality of endonucleases is used. Anyone skilled in the art will be able to find or determine a number of suitable restriction enzymes using any of many available DNA sequence analysis computer programs (for example, see the MacMolly Tetra package for Apple Macintosh, the details of which package are hereby incorporated by reference into this application, available free at the following URL or Internet link: http://www.Mologen.com). The size of the bacterial genome makes it very likely that any endonuclease will find numerous sites on the bacterial genome; statistically, a six-basepair recognition site is contained about 250 times in a one-megabasepair genome. In any event, the genomic DNA may be expected to be fragmented by shear forces during preparation, so that the additional endonuclease digestion should serve only to accelerate the subsequent exonucleolytic step.
The above-discussed embodiments of the present invention will be described further hereinbelow with reference to the accompanying examples. When the word xe2x80x9cinventionxe2x80x9d is used in this specification, the word xe2x80x9cinventionxe2x80x9d includes xe2x80x9cinventionsxe2x80x9d, that is, the plural of xe2x80x9cinventionxe2x80x9d. By stating xe2x80x9cinventionxe2x80x9d, Applicants do not in any way admit that the present application does not include more than one patentably and non-obviously distinct invention. Applicants maintain that this application may include more than one patentably and non-obviously distinct invention. Applicants hereby assert that the disclosure of this application may include more than one invention, and, in the event that there is more than one invention, that these inventions may be patentable and non-obvious one with respect to the other.